This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During every cell cycle, chromosomes must be accurately partitioned to daughter cells to prevent genomic instability and aneuploidy, a hallmark of all tumors and many birth defects. Chromosomes segregate using their kinetochores, the specialized protein structures that are assembled on centromeric DNA sequences and mediate attachment to the spindle. One hallmark of all eukaryotic kinetochores is an essential centromeric histone H3 (CenH3) variant that localizes exclusively to centromeres and replaces canonical histone H3 in centromeric nucleosomes. Because centromeric DNA sequences are not conserved, CenH3 has been proposed to be the epigenetic component that specifies the site of kinetochore assembly. Although CenH3 is an essential component of all kinetochores and is required for centromeric chromatin structure, little is known about CenH3 incorporation into centromeric DNA. We therefore propose to purify the budding yeast CenH3, Cse4, to identify interacting proteins that may regulate its exclusive deposition at the centromere. Because we previously found that Cse4 is degraded by ubiquitin-mediated proteolysis, we created a stable Cse4 protein by mutating all of the lysine residues to arginine (Cse4K16R) (Collins et al., 2004). The Cse4K16R protein is significantly stabilized allowing the accumulation of soluble Cse4 protein. Because Cse4K16R is still targeted to the centromere, it still interacts with the deposition factors that load it at the centromere. We have therefore constructed a Cse4K16R-FLAG protein and purified it by anti-FLAG immunoprecipitation (data not shown). We are currently performing a second step purification using anion exchange chromatography and propose to submit this purified protein for mass spectrometry to identify interacting proteins. We will subsequently test whether the proteins identified interact with the wildtype Cse4 protein and assist in its loading at the yeast centromere. Taken together, these studies should identify proteins that lead to Cse4 deposition at the centromere. Collins, K. A., S. Furuyama and S. Biggins. 2004. Proteolysis contributes to the exclusive centromere localization of the yeast Cse4/CENP-A histone H3 variant. Curr Biol. 14 (21) 1968-72.